Altered DNA repair pathway engagement by engineered CRISPR-Cas9 nucleases
Name
chauhan-et-al-2023-altered-dna-repair-pathway-engagement-by-engineered-crispr-cas9-nucleases.pdf
Description
Published version
Size
1.53 MB
Format
Adobe PDF
Checksum (MD5)
83866ffd845a461aca64ab265dcf65ea
Author(s)
Chauhan, Vikash P
Sharp, Phillip A
Langer, Robert
Date Issued
March 7, 2023
Journal
Proceedings of the National Academy of Sciences
Publisher
Proceedings of the National Academy of Sciences
Citation
V.P. Chauhan,P.A. Sharp, & R. Langer, Altered DNA repair pathway engagement by engineered CRISPR-Cas9 nucleases, Proc. Natl. Acad. Sci. U.S.A. 120 (11) e2300605120.
Version
Final published version
Abstract
CRISPR-Cas9 introduces targeted DNA breaks that engage competing DNA repair pathways, producing a spectrum of imprecise insertion/deletion mutations (indels) and precise templated mutations (precise edits). The relative frequencies of these pathways are thought to primarily depend on genomic sequence and cell state contexts, limiting control over mutational outcomes. Here, we report that engineered Cas9 nucleases that create different DNA break structures engage competing repair pathways at dramatically altered frequencies. We accordingly designed a Cas9 variant (vCas9) that produces breaks which suppress otherwise dominant nonhomologous end-joining (NHEJ) repair. Instead, breaks created by vCas9 are predominantly repaired by pathways utilizing homologous sequences, specifically microhomology-mediated end-joining (MMEJ) and homology-directed repair (HDR). Consequently, vCas9 enables efficient precise editing through HDR or MMEJ while suppressing indels caused by NHEJ in dividing and nondividing cells. These findings establish a paradigm of targeted nucleases custom-designed for specific mutational applications.
MIT Department
Koch Institute for Integrative Cancer Research at MIT
Massachusetts Institute of Technology. Department of Biology
Massachusetts Institute of Technology. Department of Chemical Engineering
Terms of Use
Creative Commons Attribution-NonCommercial-NoDerivatives
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1073/pnas.2300605120
